Design and Biological Evaluation of Manganese- and Ruthenium-Based Hybrid CO-RMs (HYCOs)

Article abstract of DOI:10.1002/cmdc.201900426

Interest in the therapeutic effects of carbon monoxide (CO), a product of heme degradation catalyzed by the enzyme heme oxygenase-1 (HO-1), has led to the development of CO-releasing molecules (CO-RMs) for the controlled delivery of this gas in vivo. We recently proposed conjugating a cobalt-based CO-RM with various activators of nuclear factor erythroid 2-related factor 2 (Nrf2), the transcription factor that regulates HO-1 expression, in order to exploit the beneficial effects of exogenous and endogenous CO. In this study, we describe the preparation of hybrid molecules (termed HYCOs) conjugating a fumaric acid derivative as an Nrf2 activator to a Mn- or a Ru-based CO-RM known to be pharmacologically active. With the exception of an acyl-manganese complex, these hybrids were obtained by associating the two bioactive entities by means of a linker of variable structure. X-ray diffraction analyses and preliminary biological investigations are also presented.

Full text of DOI:10.1002/cmdc.201900426

Accepted Article
Title: Recent Developments in the Design of Mn- and Ru-based Hybrid
CO-Releasing Molecules (HYCOs)
Authors: Anthony Ollivier, Roberta Foresti, Zeina El Ali, Thierry
Martens, Hiroaki Kitagishi, Roberto Motterlini, and Michael
Rivard
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Recent Developments in the Design of Mn- and Ru-based Hybrid
CO-Releasing Molecules (HYCOs)
Anthony Ollivier,[a] Roberta Foresti,[b,c] Zeina El Ali,[b,c] Thierry Martens,[a] Hiroaki Kitagishi,[d] Roberto
Motterlini,*[b,c] and Michael Rivard*[a]
oxygenase (HO)/CO pathway may set the path for drug
discovery.[9]
Abstract: The interest in the therapeutic effects of carbon monoxide
(CO), a product of the enzyme heme oxygenase-1 (HO-1), led to the
development of CO-releasing molecules (CO-RMs) for the controlled
delivery of this gas in vivo. We have recently proposed to conjugate
CO-RMs with Nrf2 activators, the transcription factor that regulates
HO-1 expression, in order to make use of the beneficial effects of
exogenous and endogenous CO. In this study we describe the
preparation of hybrid molecules conjugating a fumaric acid derivative
as Nrf2 activator with Mn- or Ru-based CO-RMs known to be
pharmacologically active. With the exception of an acyl-Mn complex,
these hybrids (HYCOs) were obtained by associating the two
bioactive entities by means of a linker of variable structure. X-ray
diffraction analyses, as well as preliminary biological evaluations, are
also presented.
Recently, we developed the concept of hybrid molecules –
termed HYCOs – by conjugating a CO-RM with various Nrf2
activators (Figure 1). Nrf2 is a transcription factor that regulates
the expression of several cytoprotective genes, including HO-1,
the inducible isoform of HO. HYCO-1 (Figure 1) and HYCO-2
produced the first examples of hybrids.[10] These hybrids resulted
from the conjugation of a cobalt-carbonyl complex with a fumaric
acid derivative as Nrf2/HO-1 activator. The choice of the latter
was justified since dimethyl fumarate is an immunomodulatory
drug clinically applied for the treatment of multiple sclerosis.[11]
Later on, and following the same idea, hybrids bearing an Fe-
carbonyl have been reported.[12]
Introduction
Carbon monoxide (CO) is interesting as a therapeutic agent due
to its importance in cell signaling and in particular for its anti-
inflammatory, anti-ischemic and vasodilatory properties.[1] Over
the last decade, the pharmacological approach to using the
therapeutic effect of CO has consisted in developing CO-
releasing molecules (CO-RMs) – mainly but not exclusively
based on carbonyl complexes[2] – capable of carrying and
delivering exogenous CO to biological systems.[3] Pioneering
pharmaceutical agents such as CORM-3 or CORM-401 (Figure
4]
1)[3b,
– respectively consisting in Ru- or Mn-carbonyls –
prompted the synthesis of new compounds aiming to obtain
better biocompatibility,[5] toxicity profile, cellular targets[2d, 6] or to
improve control in the release of CO, e.g. by enzymatic[7] (ET-
CORMs) or photochemical action[8] (PhotoCORMs). Notably, a
variety of CO-RMs have proven their therapeutic activity in
different animal models of disease confirming that the heme-
[a]
[b]
Dr. A. Ollivier, Pr. T. Martens, and Dr. M. Rivard
Université Paris Est, ICMPE (UMR 7182), CNRS, UPEC, F-94320
Thiais France
E-mail: rivard@icmpe.cnrs.fr
Dr. Roberta Foresti, Dr. Zeina El Ali, Dr. Roberto Motterlini
INSERM U955, Equipe 12, F-94000 Créteil France
E-mail: roberto.motterlin@inserm.fr
Figure 1. CO-RMs and Hybrid CO-RMs (HYCOs).
[c]
[d]
Dr. Roberta Foresti, Dr. Zeina El Ali, Dr. Roberto Motterlini
Université Paris Est, Faculté de Médecine, F-94000 Créteil France
Dr. Hiroaki Kitagishi
Department of Molecular Chemistry and Biochemistry, Faculty of
Science and Engineering, Doshisha University, Kyotanabe, Kyoto,
610-0321, Japan
In an effort to optimize the efficacy of the HYCOs, a structure-
activity study was initiated, first focusing on the type of Nrf2/HO-
1 activators to be selected.[13] This study confirmed the
interesting properties of the fumaric derivative HYCO-1, as this
hybrid revealed to be the less toxic among a series of cobalt-
based HYCOs bearing various Nrf2/HO-1 activators. This study
also showed that most of these HYCOs were capable of
Supporting information for this article is given via a link at the end of
the document.
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activating Nrf2 and simultaneously liberating CO. In this series,
HYCOs’ capability to release CO was assessed in vitro, using
typical myoglobin or hemoglobin (Hb) assays. The dual activity
of cobalt-based HYCOs was confirmed by in vivo experiments in
mice (determined by the increase of carboxyhemoglobin (HbCO)
in blood). However, an increase of intracellular CO in cultured
cells could not be demonstrated, which was attributed to the
stability of cobalt-carbonyls and their ability to release
undetectable amounts of CO.[10, 13] Consequently, the structure-
activity study next focused on the conjugation of the Nrf2/HO-1
activator previously identified to be the less toxic with CO-RMs
susceptible to deliver higher amounts of CO than cobalt-
carbonyls. This led to the preparation of HYCO-3 and HYCO-6,
two hybrids recently described deriving from fumaric acid and
bearing respectively a Mn- or a Ru-carbonyl complex as a CO-
RM (Figure 1).[14] The promising results obtained regarding their
toxicity profile, CO release and cytoprotective response,
prompted synthesizing analogs of these two compounds.
by means of a linker. While the connection of this linker to the
fumaric moiety was expected to proceed by esterification, its
coordination to the metal carbonyl could happen either with a
dithiocarbamate function as for CORM-401[15] or, alternatively,
with an amino acid as for CORM-3[16] (Figure 1). In the latter
case, the lability of the ligand prevented the formation of stable
complexes. As a result, any attempts to prepare hybrids bearing
linkers deriving from L-serine or analogs remained unsuccessful
(data not shown). Consequently, a linker with dithiocarbamate
function capable of strongly coordinating the metal carbonyls
was chosen.
Apart from the strategy allowing the conjugation of two bioactive
entities, it seemed interesting to vary the nature of the linker in
order to modulate the physicochemical properties (e.g. the
hydrosolubity) of the resulting HYCOs. This modulation was
expected by using one of the three nitrogen-containing linkers A,
B and C (Figure 1).
Using linker A led to obtaining of HYCO-3 and HYCO-6, two
hybrids bearing respectively a Mn- and a Ru-carbonyl complex.
The pharmacological evaluation of these two hybrids has
recently been published,[14] yet the detailed synthesis and full
chemical characterization of these two compounds and their
synthetic intermediates is introduced in the present article.
In brief, HYCO-3 and HYCO-6 were obtained in 3 steps from N-
methyl aminoethanol 1 (Scheme 1, pathway 1). The preparation
of the corresponding dithiocarbamate sodium salt 2 proceeded
almost quantitatively and its complexation with either
Mn2(CO)10[17] or [RuCl2(CO)3]2 gave the metal-carbonyls 3a or 3b,
in 94 and 93% yield, respectively. Reaction with ethyl fumaroyl
This paper presents the description of the detailed synthesis and
the full chemical characterization of HYCO-3, HYCO-6 and of
their analogs. The preliminary results of their biological
evaluation are also presented.
Results and Discussion
Synthesis of HYCOs
For this new series of HYCOs, the conjugation of a Mn- or a Ru-
carbonyl complex with a fumaric acid derivative was carried out
Scheme 1. Synthesis of Mn- and Ru-based HYCOs.
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chloride in the presence of DMAP as catalyst gave the expected
hybrids in 54% yield after chromatography on silica gel. It should
be pointed out that the preparation of HYCO-6 gave the first
example of a Ru-carbonyl coordinated with a dithiocarbamate
ligand. Interestingly, from this hybrid we obtained a single crystal,
suitable for an X-ray characterization (vide infra).
Following a comparable synthetic approach, a second series of
HYCOs integrating the linker B within their structure was
synthesized from hydroxyethyl piperazine 4 (Scheme 1, pathway
2). After the preparation of the dithiocarbamate salt 5 obtained
almost quantitatively from 4, complexation with MnBr(CO)5 or
[RuCl2(CO)3]2 gave the corresponding alcohols 6a and 6b,
respectively isolated in yields of 94 and 87%. Expected hybrids
were obtained after esterification. Flash chromatography on
silica gel gave HYCO-7 and HYCO-11 in yields of 45 and 40%,
respectively.
The preparation of a hybrid bearing linker C was achieved with
the synthesis of HYCO-13 (Scheme 1, pathway 3) from triazole
7 (for its preparation, see the Supporting Information). Similarly
to compounds 3a and 6a, alcohol 8 was obtained in 2 steps from
7 with an overall yield of 94%. Subsequent esterification gave
HYCO-13 isolated after flash chromatography on silica gel with a
moderate yield of 27%.
Finally, the known ability of Mn to form acyl-metal carbonyl
complexes[18] has been exploited for the preparation of the last
HYCO bearing no linker (Scheme 1, pathway 4). Reduction of
Mn2(CO)10 by a Na(Hg) amalgam and subsequent reaction with
ethyl fumaroyl chloride gave HYCO-12 with a low yield of only
5% after crystallization.
X-ray characterization of HYCO-6 and HYCO-7
Slow diffusion of petrol ether in a solution of HYCO-6 in ethyl
acetate under argon at -18 °C yielded a single crystal suitable
for X-ray diffraction analysis (Figure 2). HYCO-6 crystallized in a
monoclinic space group P 21/n characterized by cell parameters
a = 14.1997(7) Å, b = 6.0205(2) Å, c = 21.8501(9) Å;  =
90.807(2) ° and a cell volume of 1867.76(14) Å3. As for CORM-3,
the solid state structure indicated an octahedral geometry
around the Ru atom with fac-configured carbonyls, the
dithiocarbamate ligand occupying the remaining equatorial
positions. While two different equatorial Ru-CO bond lengths
were observed within CORM-3 (1.903(3) and 1.943(3) Å)[16] due
to the different nature of heteroatoms coordinated to the metallic
center, HYCO-6 exhibited two identical equatorial Ru-CO bond
lengths (1.943(3) Å, see the Supporting Information). Rather
unexpectedly, HYCO-6 crystallized in a bent conformation, the
fumaric moiety overhanging one of the two apical positions of
the organometallic complex. In the solid, the fumaric moieties of
adjacent molecules, along b axis, assembled in a plane which
interacts with an equivalent plane, along the a axis, thus forming
layers of fumaric moieties separated by layers of ruthenium
complexes (Figure 2).
Slow diffusion of pentane in a solution of HYCO-7 in diethyl
ether under argon at -18 °C gave a single crystal suitable for X-
ray diffraction analysis (Figure 3). HYCO-7 crystallized in a
3.17 Å
Figure 2. X-ray molecular representation of HYCO-6 with thermal ellipsoids set at 30% probability (left) and crystal packing (right).
monoclinic space group P 21/n with cell parameters a =
10.0693(8) Å, b = 9.8112(8) Å, c = 21.8667(17) Å;  =
96.704(3) ° and a cell volume of 2145.5(3) Å3. As for CORM-
401,[15] compound HYCO-7 presented an octahedral geometry
around the Mn atom with the dithiocarbamate ligand in
equatorial position. Compared with HYCO-6, HYCO-7 showed
shorter equatorial Metal-CO bonds (1.8172(16) or 1.8184(16) Å)
whereas axial Metal-CO bonds lengths were similar within both
complexes (1.879(4) or 1.927(15) Å for HYCO-6, 1.8496(16) and
Figure 3. X-ray molecular representation of HYCO-7 with thermal ellipsoids
set at 30% probability.
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1.8719(16) Å for HYCO-7, see the Supporting Information).
Unlike HYCO-6, HYCO-7 crystallized in a linear conformation,
the presence of the piperazine cycle preventing any folding of
the molecule.
the known stability of cobalt-carbonyls. Regarding HYCO-3 and
HYCO-6 – despite both hybrids having revealed a capability to
deliver CO to Hb in vitro – intracellular CO accumulation was
observed only with HYCO-3 but not with the Ru-based hybrid
HYCO-6 in the presence of COP-1.[14] In this case, owing this to
the stability of the metal carbonyl seemed quite unlikely in view
of the in vitro kinetic studies. In the present study, the
quantification of intracellular CO was consequently performed
using hemoCD, a recently developed porphyrin-cyclodextrin
complex that acts as a potent scavenger of CO.[21]
HemoCD was used to assess the ability of HYCO-3, HYCO-6
and HYCO-13 to deliver CO to BV2 microglia cells. As shown in
Figure 5, the Mn-based hybrid HYCO-3 produced the highest
accumulation of CO in cells in a time-dependent manner. After 3
h of exposure at 5 µM the levels of intracellular CO in the
presence of HYCO-3 remained significantly higher than in the
controls. The same applied after 24 h at 10 µM. It should be also
noted that HYCO-3 gave comparable results at a concentration
of 10 µM to CORM-401 incubated in cells at a concentration of
100 µM. This observation might be explained by HYCO-3 having
a better cell penetration than CORM-401 (the latter being a polar
compound, negatively charged at physiologic pH because of its
carboxylic acid function) and/or by CO being released from the
HYCO’s CO-RM moiety, combined with a cellular CO production
due to the fumaric moiety activating HO-1.
Detection of CO with hemoglobin assay
Kinetic studies were conducted with hemoglobin (Hb) and the
CO released from the HYCOs was evaluated by measuring the
formation of carboxyhemoglobin (HbCO) over time. With the
noticeable exception of HYCO-12, Mn-based HYCOs gave
comparable results. The half-lives of the HbCO formations were
8.6 and 11.3 min for HYCO-7 and HYCO-13 respectively,
complying with that of 11.1 min previously measured for HYCO-
3,[14] and to a lesser extent with CORM-401 (19.7 min, Figure 4).
Ru-HYCOs
Mn-HYCOs
100
100
75
75
CORM-401
CORM-401
HYCO-6
50
25
0
50
25
0
HYCO-3
HYCO-13
HYCO-12
HYCO-7
HYCO-11
0
50
100
150
0
20
40
60
80
Time / min
Time / min
Figure 4. Kinetic of CO release from HYCOs to hemoglobin.
2.5
*
2.0
1.5
1.0
0.5
1 h
3 h
24 h
These results suggest a comparable behavior in solution of Mn-
carbonyl complexes when coordinated with a dithiocarbamate
ligand. The half-life of HbCO formation dramatically increased
to 65.0 min with HYCO-12, which may be attributed to the
relative stability of this compound under the conditions of the
experiment (i.e. in an aqueous medium).
*
*
*
*
*
*
*
*
*
*
*
0.0
(M)
5
0
5
0
1
5
0
0
0
5
0
5
0
1
5
0
0
0
5
0
5
0
5
0
0
0
C
C
C
1
1
0
0
1
1
0
0
1
1
1
0
0
1
2
1
2
1
2
3
3
1
3
1
1
3
6
-
6
6
-
3
-
3
1
-
1
-
1
-
0
-
0
0
-
-
4
4
-
O
O
4
O
C
O
C
O
O
Regarding the Ru-series, the half-life measured with HYCO-11
was 1.38 min, comparable to the 3.78 min previously reported
-
O
-
O
C
O
C
C
C
C
C
M
C
Y
M
M
Y
Y
Y
H
Y
Y
Y
Y
Y
R
R
R
H
H
H
H
H
H
H
H
O
O
O
C
C
C
for HYCO-6 (Figure 4).[14] As expected, the use of
a
Figure 5. Accumulation of intracellular CO at different time points after treating
BV2 cells with HYCOs and CORM-401. Data represent the mean ± S.E.M. of
n = 3-5 independent experiments.
dithiocarbamate ligand instead of an amino acid resulted in
obtaining more stable complexes and exhibiting lower kinetics of
CO release compared to CORM-3 (half-life < 1 min).[4a] However,
the kinetics measured with the Ru-based hybrids remained
significantly faster than with their Mn-based analogs. This
suggests that under our conditions, Ru-based HYCOs are less
stable than their Mn-based counterparts, whatever the nature of
the ligand.[19] Although no clear explanation was to be found, it
also appears that using linker B yields compounds exhibiting
faster kinetics of CO release.
In accordance with results obtained with COP-1, no CO was
detected in microglia cells treated with the Ru-based analog
HYCO-6. Excluding the hypothesis of an unexpected stability of
the carbonyl complex, two possible explanations for these
findings remain: 1) the faster kinetic of CO release by HYCO-6
prevented this hybrid from effectively delivering CO into cells, as
CO may be dispersed immediately into the medium; 2) Ru-
based HYCOs have a reduced ability to penetrate cells
compared to Mn-based HYCOs.
Despite HYCO-13 displaying a kinetic of CO release similar to
that of HYCO-3 in vitro, this hybrid was markedly less effective.
Therefore, introducing the polar triazole nucleus with the linker C
did not affect the rate of CO release to Hb but significantly
diminished the ability of HYCO-13 to deliver CO into the cells.
Delivery of CO to cells
As previously mentioned, the release of CO to cells from cobalt-
based HYCOs was indirectly proven in in vivo experiments,
showing the increase of HbCO in blood. If myoglobin or Hb
assays demonstrated the capability of these hybrids to release
13]
CO in vitro,[10,
any attempts to observe an increase of
intracellular CO remained unsuccessful when using the
palladium-containing fluorescence probe COP-1,[20] even after
applying them to cells for several hours.[13] This result was
attributed to the release of tiny amounts of CO, consistent with
Conclusions
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was obtained in 97% yield as a pale-yellow powder and engaged in the
next step without further purification. 1H NMR (400 MHz, CD3OD) δ 4.27
(t, J = 6.0 Hz, 2H), 3.87 (t, J = 6.0 Hz, 2H), 3.57 (s, 3H). 13C NMR (100
MHz, CD3OD) δ 213.99, 61.14, 59.07, 44.65.
This article describes the synthesis and the full chemical
characterization of various hybrid CO-RMs (termed HYCOs)
associating a Mn- or a Ru-based CO-RM with a fumaric acid
derivative previously identified as an efficient Nrf2/HO-1
activator. The X-ray diffraction analysis of two of these
Synthesis of Mn-complex 3a. Salt
2 (87 mg, 0.50 mmol, 1.0
equiv) was added to a degassed solution of Mn(CO)5Br (137 mg,
0.50 mmol, 1.0 equiv) in methanol (8 mL). After 3.5 h of stirring at
45 °C, the reaction mixture was concentrated in vacuo. Dissolution
of the resulting solid in diethyl ether, filtration on celite and
concentration gave the expected product. Complex 3a (149 mg,
0.47 mmol) was obtained in 94% yield as a yellow oil and engaged
in the next step without further purification. 1H NMR (400 MHz,
CD3OD) δ 3.87 (t, J = 5.3 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 3.33 (s,
3H). 13C NMR (100 MHz, CD3OD) δ 209.23, 188.25, 59.94, 54.65,
38.74. 55Mn NMR (100 MHz, CD3OD) δ -1035.4. Anal. Calcd for
C8H8MnNO5S2 (317.22): C, 30.29; H, 2.54; N, 4.42. Found: C,
30.50; H, 2.54; N, 4.23.
compounds yielded the first structure of
a Ru-carbonyl
coordinated to a dithiocarbamate. The biological evaluation
confirmed the ability of these HYCOs to release CO to Hb.
However, since the Ru-based HYCOs’ kinetics of CO release
proved too fast, they are probably not suitable for delivering CO
efficiently to biological systems. In contrast, the extremely slow
kinetic observed with the acyl-Mn HYCO-12 confirmed the need
for a linker that associate a Mn-based CO-RM with a fumaric
acid derivative. The use of nitrogen containing linkers B or C did
not produce any hybrids more efficient than HYCO-3, which in
consequence appears to be the best CO-RM/linker/HO-1-
activator combination tested to date. As a dual activity molecule
that activates Nrf2 and simultaneously delivers CO, HYCO-3
appears to be an extremely promising therapeutic agent. Finally,
the results obtained with HYCO-6 clearly showed that the Hb or
myoglobin assays for measuring the kinetic of CO release in
vitro are not sufficient in determining the ability of a CO-RM
(hybridized or not) to deliver CO to cells or tissues. In view of
this work and as recently reported,[22] any satisfactory
Synthesis of Ru-complex 3b. Salt 2 (219 mg, 1.26 mmol, 1.0 equiv)
was added to a degassed suspension of [RuCl2(CO)3]2 (324 mg, 0.63
mmol, 0.5 equiv) in methanol (13 mL). After 3 h of stirring at 20 °C, the
reaction mixture was concentrated in vacuo. Dissolution of the resulting
solid in ethyl acetate, filtration on celite and concentration gave the
expected product. Complex 3b (435 mg, 1.17 mmol) was obtained in
93% yield as a pale-yellow solid and engaged in the next step without
further purification. 1H NMR (400 MHz, CD3OD) δ 3.96-3.77 (m, 4H),
3.37 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 210.27, 188.95, 188.72,
59.81, 55.03, 38.86.
characterization of
a CO-based therapeutic agent clearly
requires the combined assessment of CO release in vitro
(myoglobin or hemoglobin assay) as well as CO accumulation in
cells (COP-1 or hemoCD assay).
Synthesis of HYCO-3. Ethyl fumaroyl chloride (300 mg, 1.85 mmol, 1.5
equiv) and 4-DMAP (225 mg, 1.85 mmol, 1.5 equiv) were successively
added to a solution of 3a (390 mg, 1.23 mmol, 1.0 equiv) in anhydrous
and degassed dichloromethane (20 mL). After 18 h of stirring at 40 °C,
the reaction mixture was concentrated in vacuo. The resulting crude
product was purified on silica gel (cyclohexane/ethyl acetate, 70/30 +
0.1 % triethylamine) and dried overnight with a vane pump. HYCO-3 (295
mg, 0.67 mmol) was obtained in 54% yield as a yellow solid. 1H NMR
(400 MHz, C6D6) δ 6.93 (d, J = 15.8 Hz, 2H), 6.85 (d, J = 15.8 Hz, 2H),
3.91 (q, J = 7.1 Hz, 2H), 3.67 (t, J = 5.2 Hz, 2H), 3.04 (t, J = 5.2 Hz, 2H),
2.36 (s, 3H), 0.89 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
210.05, 164.67, 134.91, 132.36, 61.50, 61.42, 49.78, 37.76, 14.07. 55Mn
NMR (100 MHz, CD3OD) δ -1033.40. Anal. Calcd for C14H14MnNO8S2
(443.33): C, 37.93; H, 3.18; N, 3.16. Found: C, 37.87; H, 3.07; N, 3.21.
Experimental Section
Chemistry
All reactions were performed under
a dry, anaerobic argon
atmosphere. Mn2(CO)10, [RuCl2(CO)3]2 and all other reagents
commercially available were used without further purification.
Mn(CO)5Br[17] and ethyl fumaroyl chloride[23] were prepared as
previously described. Silica gel 60A (SDS) was used for column
chromatography. Merk silica gel plates 60 F254 (0.25 mm thick) were
used for analytical thin layer chromatography (TLC). TLC were
observed under UV at 254 nm and revealed by heating after
Synthesis of HYCO-6.
Ethyl fumaroyl chloride (285 mg, 1.76 mmol,
1.5 equiv) and 4-DMAP (215 mg, 1.76 mmol, 1.5 equiv) were
successively added to a solution of 3b (435 mg, 1.17 mmol, 1.0 equiv) in
anhydrous and degassed dichloromethane (16 mL). After 17 h of stirring
at 40 °C, the reaction mixture was concentrated in vacuo. The resulting
crude product was purified on silica gel (cyclohexane/ethyl acetate,
60/40) and dried overnight with a vane pump. HYCO-6 (312 mg, 0.63
mmol) was obtained in 50% yield as a white solid. 1H NMR (400 MHz,
CDCl3) δ 6.89 (d, J = 15.8 Hz, 1H), 6.85 (d, J = 15.8 Hz, 1H), 4.48 (m,
2H), 4.30 (m, 1H), 4.26 (q, J = 7.2 Hz, 2H), 3.80 (m, 1H), 3.35 (s, 3H),
1.32 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 211.09, 186.98,
186.67, 186.66, 164.59, 164.47, 135.07, 132.17, 61.51, 61.13, 50.09,
37.92, 14.05. Anal. Calcd for C13H14ClNO7RuS2 (496.91): C, 31.42; H,
2.84; N, 2.82. Found: C, 31.49; H, 2.80; N, 2.79.
1
treatment with KMnO4. H, 13C and 55Mn spectra were collected in
CD3OD, CDCl3 or C6D6 at 20 °C and were recorded on a Bruker
Avance-II 400 MHz spectrometer. Chemical shifts are reported in
parts per million (ppm) and referenced to the residual solvent peak:
δ1H(CD3OD) = 3.31 ppm, δ13C(CD3OD) = 49.00 ppm, δ1H(CDCl3) =
7.26 ppm, δ13C(CDCl3) = 77.00 ppm, δ1H(C6D6) = 7.16 ppm,
δ13C(C6D6) = 128.06 ppm, J values are given in Hz. A saturated
solution of KMnO4 in D2O was used as reference for 55Mn NMR
experiments. Elemental analyses were performed on a Perkin-
Elmer 2400 apparatus at the “Institut de Chimie des Substances
Naturelles” (Gif sur Yvette, France).
Synthesis of dithiocarbamate salt 2. 2-(methylamino)ethanol 1 (400 µL,
5.00 mmol, 1.0 equiv) was added dropwise at 0 °C to a solution of carbon
disulfide (452 µL, 7.50 mmol, 1.5 equiv) in anhydrous tetrahydrofuran (10
mL). After 5 min of stirring at 0 °C, sodium hydride (60% dispersion in
mineral oil, 200 mg, 5.00 mmol, 1.0 equiv) was added portionwise. After
30 min of stirring at 0 °C, the reaction mixture was concentrated in vacuo.
After trituration with cyclohexane to remove the mineral oil, the resulting
solid was dried overnight with a vane pump. Salt 2 (842 mg, 4.9 mmol)
Synthesis of dithiocarbamate salt 5. Carbon disulfide (452 µL, 7.50
mmol, 1.5 equiv) was added dropwise at 0 °C to a solution of 1-(2-
hydroxyethyl)piperazine 4 (651 mg, 5.00 mmol, 1.0 equiv) in anhydrous
tetrahydrofuran (15 mL). After 15 min of stirring at 0 °C, sodium hydride
(60% dispersion in mineral oil, 200 mg, 5.00 mmol, 1.0 equiv) was added
portionwise. After 40 min of stirring at 0 °C, the reaction mixture was
concentrated in vacuo. After trituration with cyclohexane to remove the
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10.1002/cmdc.201900426
ChemMedChem
FULL PAPER
mineral oil, the resulting solid was dried overnight with a vane pump. Salt
5 (1.10 g, 4.82 mmol) was obtained in 96% yield as a pale-yellow powder
and engaged in the next step without further purification.1H NMR (400
MHz, CD3OD) δ 4.46 (t, J = 4.8 Hz, 4H), 3.71 (t, J = 5.9 Hz, 2H,), 2.55 (t,
J = 5.1 Hz, 6H). 13C NMR (100 MHz, CD3OD) δ 60.98, 59.80, 54.35,
51.20.
mixture of methanol/cyclohexane (1/1). After decantation, the methanol
layer was washed by cyclohexane, concentrated in vacuo and the
resulting solid was dried overnight with a vane pump. The expected
dithiocarbamate salt (395 mg, 1.55 mmol) was obtained in 95% yield as a
brown solid and engaged in the next step without further purification. 1H
NMR (400 MHz, CD3OD) δ 7.93 (s, 1H), 4.68 (s, 2H), 4.51 (t, J = 6.2 Hz,
2H), 3.04 (t, J = 6.2 Hz, 2H), 2.39 (s, 3H). 13C NMR (100 MHz, CD3OD
δ 149.10, 124.51, 56.48, 51.76, 50.50, 35.74.
)
Synthesis of Mn-complex 6a. Mn(CO)5Br (346 mg, 1.26 mmol, 1.0
equiv) was added to a degassed solution of 5 (318 mg, 1.39 mmol, 1.1
equiv) in methanol (20 mL). After 3 h of stirring at 45 °C, the reaction
mixture was concentrated in vacuo. Dissolution of the resulting solid in
diethyl ether, filtration on celite and concentration gave the expected
product. Complex 6a (443 mg, 0.32 mmol) was obtained in 94% yield as
a yellow oil and engaged in the next step without further purification. 1H
NMR (400 MHz, CD3OD) δ 3.91 (s, 4H), 3.68 (s, 2H), 2.58 (s, 6H). 13C
NMR (100 MHz, CD3OD) δ 207.67, 60.60, 59.79, 53.34, 46.86. 55Mn
NMR (100 MHz, CD3OD) δ -1025.7. Anal. Calcd for C11H13MnN2O5S2
(372.30): C, 35.49; H, 3.52; N, 7.52. Found: C, 35.64; H, 3.71; N, 7.10.
2) Mn(CO)5Br (432 mg, 1.55 mmol, 1.0 equiv) was added to a degassed
solution of dithiocarbamate salt (394 mg, 1.55 mmol, 1.0 equiv) in
methanol (27 mL). After 3 h of stirring at 45 °C, the reaction mixture was
concentrated in vacuo. Dissolution of the resulting solid in ethyl acetate,
filtration on celite and concentration gave the expected product. Complex
8 (613 mg, 1.54 mmol) was obtained in 99% yield as a yellow solid and
engaged in the next step without further purification. 1H NMR (400 MHz,
CD3OD) δ 7.93 (s, 1H), 4.75 (t, J = 5.7 Hz, 2H), 4.67 (s, 2H), 4.26 (t, J =
5.6 Hz, 2H), 3.19 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 210.99, 177.03,
149.30, 124.76, 56.50, 51.63, 47.86, 37.31. 55Mn NMR (100 MHz,
CD3OD) δ 1019.2.
Synthesis of Ru-complex 6b. Salt 5 (228 mg, 1.00 mmol, 1.0 equiv)
was added to a degassed suspension of [RuCl2(CO)3]2 (256 mg, 0.50
mmol, 0.5 equiv) in methanol (10 mL). After 4 h of stirring at 20 °C, the
reaction mixture was concentrated in vacuo. Dissolution of the resulting
solid in ethyl acetate, filtration on celite and concentration gave the
expected product. Complex 6b (370 mg, 0.87 mmol) was obtained in
87% yield as a pale-yellow solid and engaged in the next step without
further purification. 1H NMR (400 MHz, CD3OD) δ 3.96 (d, J = 5.0 Hz,
4H), 3.72 (t, J = 5.6 Hz, 2H), 2.76 (s, 4H), 2.68 (s, 2H). 13C NMR (100
MHz, CD3OD) δ 188.76, 188.59, 60.50, 59.41, 52.98, 47.00.
Synthesis of HYCO-13. Ethyl fumaroyl chloride (374 mg, 2.31 mmol,
1.5 equiv) and 4-DMAP (282 mg, 2.31 mmol, 1.5 equiv) were
successively added to a solution of 8 (613 mg, 1.54 mmol, 1.0 equiv) in
anhydrous and degassed dichloromethane (20 mL). After 16 h of stirring
at 40 °C, the reaction mixture was concentrated in vacuo. The resulting
crude product was purified on silica gel (cyclohexane/ethyl acetate, 40/60
+ 0.1% triethylamine) and dried overnight with a vane pump. HYCO-13
(193 mg, 0.35 mmol) was obtained in 27% yield as a yellow solid. 1H
NMR (400 MHz, CDCl3) δ 6.96 (d, J = 16.0 Hz, 1H), 6.91 (d, J = 15.9 Hz,
1H), 6.81 (s, 1H), 5.12 (s, 2H), 3.86 (q, J = 7.2 Hz, 2H), 3.53 (t, J = 6.2
Synthesis of HYCO-7. Ethyl fumaroyl chloride (162 mg, 1.00 mmol,
1.5 equiv) and 4-DMAP (122 mg, 1.00 mmol, 1.5 equiv) were
successively added to a solution of 6a (247 mg, 0.66 mmol, 1.0 equiv) in
anhydrous and degassed dichloromethane (9 mL). After 18 h of stirring at
40 °C, the reaction mixture was concentrated in vacuo. The resulting
crude product was purified on silica gel (cyclohexane/ethyl acetate, 70/30
+ 0.1 % triethylamine) and dried overnight with a vane pump. HYCO-7
(151 mg, 0.30 mmol) was obtained in 45% yield as a yellow solid. 1H
NMR (400 MHz, C6D6) δ 6.96 (s, 2H), 3.88 (t, J = 7.0 Hz, 2H), 3.79 (t, J =
5.7 Hz, 2H), 3.33 (t, J = 5.0 Hz, 4H), 1.87 (t, J = 5.7 Hz, 2H), 1.65 (t, J =
5.0 Hz, 4H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, C6D6) δ 206.93,
164.55, 134.27, 133.33, 61.82, 61.23, 55.78, 51.54, 45.87, 13.95. 55Mn
NMR (100 MHz, CD3OD) δ -1028.42. Anal. Calcd for C17H19MnN2O8S2
(498.41): C, 40.97; H, 3.84; N, 5.62. Found: C, 41.08; H, 4.06; N, 5.73.
Hz, 2H), 3.11 (t, J = 6.1 Hz, 2H), 2.14 (s, 3H), 0.85 (t, J = 7.2 Hz, 3H). 13
C
NMR (101 MHz, CD3OD) δ 210.96, 166.74, 166.21, 134.71, 134.30,
134.12, 124.75, 62.44, 51.62, 47.84, 37.29, 14.33. 55Mn NMR (100 MHz,
CD3OD) δ -1005.62. Anal. Calcd for C17H17MnN4O8S2 (524.41): C,
38.94; H, 3.27; N, 10.68. Found: C, 38.65; H, 3.35; N, 10.49.
Synthesis of HYCO-12. In a Schlenk tube at 20 °C, sodium (30 mg,
1.28 mmol, 2.8 equiv) was added portionwise to mercury (240 µL) under
a vigorous stirring. After disappearance of the sodium, anhydrous
tetrahydrofurane (5 mL) was introduced, followed by the addition of
Mn2(CO)10 (179 mg, 0.46 mmol, 1.0 equiv). After 3 h of vigorous stirring
at 20 °C and 10 min of decantation, the upper layer was transferred via a
cannula into another Schlenk tube and cooled to -78 °C. Then, ethyl
fumaroyl chloride (242 mg, 1.00 mmol, 2.2 equiv) was added dropwise.
After 1 h of stirring at -78 °C and additional 18 h at 20 °C, the mixture
was concentrated in vacuo. The crude was dissolved in hexane (6 mL),
stirred for 30 min at 20 °C and filtered on a fritted glass. The filtrate was
stocked at -18 °C for 18 h. The precipitate was collected and dried with a
vane pump. HYCO-12 (29 mg, 0.09 mmol) was obtained in 5% yield as a
red-orange solid. 1H NMR (400 MHz, C6D6) δ 7.40 (d, J = 15.5 Hz, 1H),
5.87 (d, J = 15.3 Hz, 1H), 3.90 (q, J = 7.2 Hz, 2H), 0.87 (t, J = 7.2 Hz, 3H).
Anal. Calcd for C11H7MnO8 (322.11): C, 41.02; H, 2.19. Found: C, 40.76;
H, 2.50.
Synthesis of HYCO-11. Ethyl fumaroyl chloride (211 mg, 1.30 mmol,
1.5 equiv) and 4-DMAP (159 mg, 1.30 mmol, 1.5 equiv) were added to a
solution of 6b (370 mg, 0.87 mmol, 1.0 equiv) in anhydrous and
degassed dichloromethane (11 mL). After 16 h of stirring at 40 °C, the
reaction mixture was concentrated in vacuo. The resulting crude product
was purified on silica gel (cyclohexane/ethyl acetate, 50/50) and dried
overnight with a vane pump. HYCO-11 (193 mg, 0.35 mmol) was
obtained in 40% yield as a white solid. 1H NMR (400 MHz, CDCl3) δ 6.87
(s, 2H), 4.34 (t, J = 5.6 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 3.88 (t, J = 4.9
Hz, 4H), 2.75 (t, J = 5.5 Hz, 2H), 2.65 (m, 4H), 1.33 (t, J = 7.1 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ 186.78, 164.82, 134.26, 133.09, 133.07,
62.09, 61.47, 56.06, 51.88, 46.48, 14.11. Anal. Calcd for
C16H19ClN2O7RuS2 (551.98): C, 34.81; H, 3.47; N, 5.08. Found: C, 35.04;
H, 3.59; N, 4.91.
X-ray crystal structure determination
A single crystal of each compound was selected, mounted onto a
cryoloop and transferred into a cold nitrogen gas stream. Intensity data
were collected either with a Bruker Kappa-APEXII diffractometer using
graphite-monochromated Mo-Kα radiation (HYCO-6) or with a Bruker
Synthesis of Mn-complex 8.
QUAZAR diffractometer using
a multi-layer mirror-focused Cu-Kα
1) Carbon disulfide (148 µL, 2.45 mmol, 1.5 equiv) was added dropwise
at 0 °C to a solution of triazole 7 (255 mg, 1.63 mmol, 1.0 equiv) in
anhydrous tetrahydrofuran (17 mL). After 10 min of stirring, sodium
hydride (60% dispersion in mineral oil, 65 mg, 1.63 mmol, 1.0 equiv) was
added portionwise. After 1 h of stirring at 0 °C, the reaction mixture was
concentrated in vacuo. The resulting crude product was dissolved in a
radiation (HYCO-7). Data collection was performed with the Bruker
APEXII suite. Unit-cell parameters determination, integration and data
reduction were carried out with SAINT program. SADABS was used for
scaling and absorption corrections. The structures were solved with
SHELXT-2014[24] and refined by full-matrix least-squares methods with
SHELXL-2014[25] using the Olex2 software package[26] (HYCO-6) or the
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10.1002/cmdc.201900426
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FULL PAPER
WinGX suite[27] (HYCO-7). All non-hydrogen atoms were refined
anisotropically. In HYCO-7, axial Cl and CO ligands are disordered over
two positions. Site occupation factors were refined and converged to a
85/15 ratio. CCDC 1864086 and 1864090 contain the supplementary
crystallographic data for this paper.
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Assessment of CO release
The release of CO from HYCOs was assessed spectrophotometrically by
measuring the conversion of deoxyhemoglobin to carboxyhemoglobin
(HbCO) using a modification of a method previously described.[28] Briefly,
fresh mouse blood (0.5 mL) was initially collected in a tube containing
10 µL EDTA (10% solution) as anticoagulant. Five microliters of blood
were transferred to a sealed cuvette containing a small magnetic bar and
4.5 mL of tris(hydroxymethyl)aminomethane solution (20 mM) previously
deoxygenated with sodium dithionite. The solution was gently mixed on a
magnetic stirrer for 10 min and absorbance spectra between 400 and
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500 nm were subsequently recorded over time using
a JASCO
spectrophotometer after addition of HYCOs (5.5 µM final concentration).
The half-life of HbCO formation was calculated through the best fitting of
the kinetic data.
Assessment of CO accumulation in BV2 microglia cells
BV2 mouse microglial cells were kindly donated by Professor Rosario
Donato (University of Perugia) and grown in RPMI-1640 containing
glucose and supplemented with L-glutamine, as previously reported.[29]
hemoCD, a specific scavenger of CO, was synthesized as previously
described.[21a] Cells (2x106 per well in 6-well dishes) were incubated with
HYCOs (5 and 10 µM) for 1, 3 and 24 h. At the end of the incubation,
hemoCD (5 µM final concentration in PBS) was added to each well and
cells were then collected, counted and disrupted by sonication. Samples
were filtered to remove insoluble materials, and the clear cell lysate
(filtrate) was measured by UV−vis absorption spectroscopy as previously
described.[21b] CO content was calculated as nanomoles/1x106 cells.
CORM-401 (100 and 200 µM) was used as a positive control for CO
accumulation in cells.[30]
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Acknowledgements
This work was supported by an International Grant from the
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Agence Nationale de la Recherche (MITO−CO), by
a
Multidisciplinary Grant from the UPEC (CHORM) and by a
maturation project from the SATT IDF-Innov (HYCO). The
authors thank Christine Gaillet for implementing the 55Mn-NMR
experiment and the ICMPE for providing funds. Mr Jérémy Forté
and Mrs Lise-Marie Chamoreau are gratefully acknowledged for
the X-ray analysis of HYCO-6 and HYCO-7 and for their helpful
comments. The authors also wish to thank Tanya Farthing for
her help.
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Keywords: Biological Activity • Drug Design • Medicinal
Chemistry • Manganese • Ruthenium
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Entry for the Table of Contents
FULL PAPER
HYCOs are hybrid compounds
consisting in an Nrf2/HO-1 activator
conjugated to a CO-Releasing
Molecule (CO-RM). This study reports
on the structure-activity relationship of
various HYCOs deriving from fumaric
acid and bearing Mn- or Ru-based
CO-RMs. Their synthesis and
Anthony Ollivier, Roberta Foresti, Zeina
El Ali, Thierry Martens, Hiroaki
Kitagishi, Roberto Motterlini,* and
Michael Rivard*
Page No. – Page No.
Recent Developments in the Design
of Mn- and Ru-based Hybrid CO-
Releasing Molecules (HYCOs)
biological activities are discussed.
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